The role of palbociclib on the alterations in CDKN2, CCNE1, E2F3, MDM2 expressions as target genes of miR-141

Introduction According to WHO, Breast cancer is widely considered to be the first or second cause of cancer-related death almost universally. Cell cycle disruption, either in the form of uncontrolled expression of cyclins or because of the suspension in negative regulatory proteins (CDK inhibitors), was found to cause breast cancer. Palbociclib as specific CDK4/6 inhibitor is used for the treatment of ER+ metastatic cancers. In this study, we are looking to investigate the effect of palbociclib on breast cancer cells and evaluate the changes in the expression of some genes involved in the cell cycle as target genes of miR-141 after treatment with this drug. We used MCF7 as functional estrogen and non-invasive and MDA-MB-231 cell lines as triple-negative type of breast cancer and a model for more aggressive. Method & materials MCF7 and MDA-MB-231 cell lines were cultured in DMEM medium. After counting cells and measuring viability, Palbociclib was administered at varying doses using the IC50 obtained from MTT, with the treatment given at two time points of 24 and 72 hours. RNA was extracted from untreated and treated cells and RNAs were converted to cDNA in the end. Gene expression changes were investigated by real-time PCR. Data management and analysis were conducted using GraphPad Prism 5.01 software. Result and conclusion Among investigated genes, E2F3 gene was not significantly affected by Palbociclib in any of cell lines and time points. Besides, the expression of CCNE1 gene was significantly suppressed. It seems this drug was unable to reduce the expression of MDM2 gene significantly in triple negative (MDA-MB-231) cancer cells; however, a decrease was observed in luminal A (MCF-7) cells. CDKN2A and miR-141 genes expression increased significantly after treatment which can be aligned with palbociclib in proliferation inhibition.


Introduction
According to the World Health Organization (WHO), breast cancer is one of the leading causes of cancer-related deaths worldwide.It affects 2.3 million people each year, making it the most common cancer among adults.Breast cancer is categorized into four subtypes based on the presence of estrogen receptor (ER), progesterone receptor (PR), and HER-2: luminal A, luminal B, HER-2 positive, and triple-negative [1].The most prevalent subtype is luminal A, which is characterized by being ER-positive (ER + ), PR-positive (PR + ), and HER-2 negative.Luminal A breast cancer generally has a good prognosis.While endocrine therapy is effective for treating luminal A breast cancer [2], chemotherapy is the only effective treatment for triple-negative breast cancer, which is known for its aggressive nature and high metastasis rate [3,4].cell lines such as MCF-7 and MDA-MB-231 are commonly used to represent these cancer subtypes in research studies [5].
Cell cycle disruption, either through uncontrolled expression of cyclins or the suspension in negative regulatory proteins(CDK inhibitors), was found to cause breast cancer [6,7].Retinoblastoma phosphorylation by the CDK4/6 cyclin complex leads to its dissociation from the E2F transcription factor, promoting cell cycle progression [8].However, this process may be downregulated by p21 and p27 known as CDK inhibitors from the CIP-KIP family, or by P16 member of the INK family [9].Moreover, by inhibiting the activity of P53 [10], MDM2 can be downregulated by P14, which is encoded by CDKN2A [11].In this context, a class of noncoding RNAs, named microRNAs play an important role in cancer biology by cell cycle regulation, proliferation, Metastasis and apoptosis [12,13].For example, the miR-141/200a cluster has been found to reduce retinoblastoma protein phosphorylation, leading to a decline in the G1/S transition phase.Meanwhile, overexpression of this microRNA further promotes CDK inhibitor proliferation and G1 arrest [14].Therefore, downregulation of miR-141 in breast cancer is significant [15].The specified genes in this study including CCNE1, CDKN2A, E2F3 and MDM2, are targets of this microRNA based on databases such as Mirbase and Mirwalk.
Palbociclib, Ribociclib and Abemaciclib, as specific CDK4/6 inhibitors, obtained FDA approval for the treatment of ER-positive metastatic cancers [16], which combined with endocrine agents like fulvestrant and letrozole, may improve survival rates [17].Evidence suggests that palbociclib, in the presence of functional retinoblastoma protein binds to the ATP-binding pocket of CDK4/6 preventing the function of CDK4/6/cyclin D Complex [18].As a consequence of this action, Rb phosphorylation is stopped and cell cycle progression from G1 to S is blocked.following palbociclib treatment, a decrease in P107, accumulation of cyclin D1 & E1 and decrease in E2F-regulated protein occur [19].
From a clinical perspective, in addition to conventional methods of breast cancer treatment such as chemotherapy drugs and hormone therapy, immunotherapy drugs can also have a promising future [20,21].Nevertheless, treatment results are often hindered by drug resistance and lack of reliable biomarkers.Therefore, a better understanding of these potential mechanisms might have gone some way toward dealing with breast cancer [22].
In this study, we performed experiments on MDA-MB-231 and MCF-7 cell lines to evaluate the effects of Palbociclib on cell growth and to analyze the changes in the expression of genes involved in the cell cycle and miR-141.

Cell lines and reagents
The MDA-MB-231 and MCF-7 cell lines used in this study were obtained from the Iranian Biological Resource Center.The cells were cultured in Dulbecco's Modified Eagle Medium/ F12 (DMEM, BioIdea) with 4.5 g/L glucose L-glutamine, supplemented with 10% fetal bovine serum (FBS, BioIdea), 100 U/mL penicillin (BioIdea, Iran) and 0.1 mg/mL streptomycin (BioIdea, Iran).The cells were maintained in a humidified incubator at 37˚C with 5% CO 2 .Once the cells reached appropriate density, they were washed with Phosphate-buffered saline (PBS) before subculturing with phenol-free trypsin/ethylene diamine tetra acetic acid (EDTA 0.25%).Palbociclib, which was purchased from MedChem Express (MCE, USA), was dissolved in 1ml sterile dimethyl sulfoxide (DMSO, DNA biotech, Iran) and stored at -20˚C.

MTT cell viability assay
Breast cancer cells were seeded at a concentration of 1×10^4 cells/mL in 100 μL per well in a 96-well plate and were incubated overnight at 37˚C.The study aimed to determine the halfmaximal inhibitory concentration (IC50) of palbociclib.After 24 hours, cells were treated with gradual doses of the drug and analyzed at 24, 48 and 72 hours.Then, 100 μL of MTT was added to each well and incubated for 4 hours.The fluorescence absorbance was measured at a wavelength of 530/590 nm using a DANA microplate ELIZA reader (Iran).

RNA extraction and qRT-PCR
To determine the impact of drugs on the expression of target genes, 3 x 10^5 cells were seeded in 6-well culture plates.The next day, the cell lines were treated with palbociclib at concentrations corresponding to the IC50 obtained from the viability test.The concentrations were 18 μM in MDA-MB-231 cells and 49 μM in MCF-7 cells.Treatment was performed at two time points, 24 and 72 hours.After treatment, cells were collected from each plate in a fresh tube and stored at -80 c .Experiments for each control and treated group were conducted in triplicate.
According to the manufacturer's instructions, total RNA was extracted using Trizol reagent (Yektatajhiz, Iran).The RNA quality and purity of each sample were validated using a Nano-Drop spectrophotometer at a 260/280 nm ratio (NanoDrop Technologies, Thermo Fisher Scientific, Inc.).The RNA was then converted into cDNA using 2 μl of random hexamer primers in a microtube containing 2 μg of RNA at a total volume of 11.4 μl using Notarkib kit (Iran).Due to the short sequences of miRNAs, cDNA synthesis of miR-141 was carried out using stem-loop specific reverse transcriptase primers.The expression level of miR-141 was quantified using specified primers followed by qRT-PCR analysis.Similarly, quantitative Real-time PCR was also performed for E2F3, CDKN2A, CCNE1, and MDM2 genes in both transfected and untreated cells using the SYBR Green master mix kit (Qiagen, Iran).Primers were designed using the primer blast site on the NCBI website and AllelID software.Also, in order to check secondary structures such as double-stranded and hairpin structures, primer sequences were checked with AllelID and Primer3.Primers were purchased from Metabion (Germany).Table 1 presents an overview of the forward (F) and reverse (R) primer sequences of miR-141 (NR_029682.1),E2F3 (NM_001949.5),CDKN2A (NM_000077.5),CCNE1 (NM_001238.4), and MDM2 (NM_002392.6)used in real-time PCR.The U6 and GAPDH mRNA genes were considered as the internal standard control gene by specific primers for miR-141 and target genes, respectively.Analysis for gene expression patterns of treated and untreated cells was performed with the 2 -ΔΔCT method.

Bioinformatics
MicroRNA target prediction: Different microRNA databases including MirBase, miRWalk, and miRanda, were investigated to determine whether the CCNE1, E2F3, CDKN2A, and MDM2 as CDK4/6/cyclin dependent are the direct targets of miR-141.GeneMANIA is a fast gene network construction and function prediction tool.The results were subsequently used to predict the interactions between TP53 and gene targets of miR-141.

Statistical analysis
GraphPad Prism 9.0.0 software was used for data management and analysis.For determining the significance of differences, two-way ANOVA analysis of variance followed by Tukey's comparison test was performed, and p-values less than 0.05 were considered statistically significant.P-values of less than 0.05, less than 0.01, and less than 0.001 were marked by *, ** and ***, respectively.

Predicted target genes of miR-141
Based on searches of online databases such as TargetScan, miRWalk, miRDB, and GeneMA-NIA, we selected MDM2, CDKN2A, CCNE1, and E2F3 as genes involved in the cell cycle and targets of miR-141 (Fig 1).

Palbociclib inhibited cell viability and proliferation of breast cancer cells
The results shown in (Fig 2) indicate that the cell viability of both MCF-7 and MDA-MD-231 cell lines decreased significantly after treatment with Palbociclib in a dose and time-dependent manner compared to the untreated control cells.This data shows that palbociclib further inhibits cell proliferation by decreasing the IC50 value over different exposure periods (24-72 hours).

Gene expression profile in palbociclib-treated breast cancer cell lines
In order to gain a detailed understanding of the effect of palbociclib on the CDK4/6 signaling pathway, expression levels of E2F3, CCNE1, CDKN2A, MDM2 genes, and miR141 at 24 and 72 hours were measured after treatment on MCF-7 and MDA-MB-231 cell lines.These cell lines represent ER +, HER2 +, and triple-negative breast cancer cells, respectively.The expression of genes at mRNA levels was investigated via qRT-PCR.Relative quantitation of genes was normalized to GAPDH (and U6 against miR-141) as a housekeeping gene.
As shown in the bar chart (Fig 3), the expression levels of CDKN2A and miR-141 in both cell lines and time points were significantly increased.In contrast, CCNE1 and MDM2 gene expression tended to decrease after palbociclib treatment, except for MDM2 gene, which was not significantly affected in MDA-MB-231 cell line.The MDM2 gene in MCF-7 cell line seems to have a greater decrease in gene expression at 24h compared to the other time point.Also, the analysis did not reveal any significant differences in E2F3 gene between two cell lines and time points of 24 and 72 hours after treatment.
As observed in (Fig 3E), palbociclib induced a significant increase in the expression of miR-141 in the MDA-MB-231 cell line at 24h (1.6 fold) more than at 72h (0.5 fold).Consequently, this drug had a greater increasing effect on the MDA-MB-231 cell line.The CDKN2A gene in the MDA-MB 231 cell line showed the most increased expression among genes which was 1.35 fold at 72h and 2.61 fold at 24h compared to control.In addition, the miR-141 gene had a higher expression level in the MCF-7 cell line.The changes in the CCNE1 gene expression in each time point and cell lines equally showed a significant decrease of approximately 0.4 fold compared to the control.

Discussion
This research has identified dose and time-dependent decreases in cell viability in both cell lines.The qRT-PCR results showed a significant increase in the expression of miR-141, a single-strand RNA that regulates gene expression at the post-transcriptional level, after palbociclib treatment in both cancer types.Among the target genes of this miRNA which was investigated in this study, E2F3, CCNE1 and MDM2 showed decreased expression due to palbociclib.However, the E2F3 gene was not significantly affected by drug in any of the cell lines and time points.Reducing the expression of these genes leads to cell cycle arrest and prevents cancer cell growth.Conversely, the expression of the CCNE1 gene as the cell cycle promoter was significantly suppressed.Palbociclib did not significantly reduce the expression of the MDM2 gene in triple negative (MDA-MB-231) cancer cells; however, a decrease was observed in luminal A (MCF-7) cells.As expected, CDKN2A gene expression as a tumor suppressor gene in both types of cancer increased significantly after treatment which can be aligned with palbociclib in proliferation inhibition.
These results support previous research which found that in MDA-MB-231 cells treated with palbociclib, genes that contributed to cell cycle upregulation and persistence were significantly suppressed, Instead, genes promoting cell cycle arrest showed increased expression [23].In a study conducted by Ping Li. et al, it has been identified that compared to surrounding intact tissues, miR-141 is downregulated in breast tumor tissues, besides overexpression of miR-141 can disrupt cell proliferation, migration and invasion [15].As mentioned above, miR-141 expression increased after treatment.Collectively, these results suggest that the rise in miR-141 may have contributed to the increase inhibition of cell growth in line with palbociclib.
In the cell cycle cascade, CDK4/6-cyclin D complex has been confirmed as an E2F activator by phosphorylating Rb, enabling the synthesis of S phase initiator proteins and conducts positive feedback for E2F gene expression proliferation [24].Out of E2F family, E2F3, along with E2F1 and E2F2, typically binds to pRb and controlled through it.While E2F4 and E2F5 are mostly controlled through P107 and P130.Phosphorylation of these regulatory proteins leads to the activation of their target genes [25].The effectiveness of CDK4/6 inhibitors on Rb phosphorylation has been demonstrated in a report by Finn et al. [26].Also, a direct effect on reducing Rb expression was noticed in the study of Choupani et al. [27].Michaloglou et al. pointed out that Palbociclib can inhibit the function of E2F by cooperating with mTORC1/2 inhibitor (Vistusertib), which has been shown to alter protein levels in the MCF-7 cell line [28].It is also stated that the E2F3 expression at the protein and mRNA level was significantly downregulated in gastric cancer cells after miR-141 transfection [29].As regards the qRT-PCR results, the expression level of E2F3 was not significantly affected after treatment with palbociclib.It may have provided insights into the theory that the observed nonsignificant result may differ at the protein level.
Contrary to ER-negative cell lines such as MDA-MB-231, all ER-positive cell lines such as MCF-7 express higher levels of MDM2 mRNA [30].Based on real-time PCR results on MDM2 gene expression, consistent with the report mentioned, palbociclib seems to considerably reduce MDM2 expression in ER+ luminal A cell line.Regarding the inhibitory effect of CDKN2A on MDM2 through the ARF protein (p14arf) in the cell cycle.according to the increased expression of CDKN2A after treatment, it can be assumed that CDKN2A works aligned with palbociclib to inhibit MDM2 at the protein level.
Due to the role of CCNE1 in cell cycle progression, boosting the CCNE1 expression in cancer cells is predictable.Turner et al. used the PALOMA-3 study to assess palbociclib's efficacy by CCNE1 mRNA expression, which confirmed the prognostic role of CCNE1 in metastatic cancer.In this way, the lower expression of CCNE1 has been certified to be associated with the progression of the palbociclib potency [31].This study provides evidence of a significant reduction of CCNE1 expression in both cancer cell lines.As well as drug impression on mRNA level, according to cyclin E/CDK2 settlement at the MDM2 downstream and inhibitory role of MDM2 on P53 [32,33], it can be concluded that palbociclib reduces MDM2 expression, which causes P53 enhancement and organized inhibitory effect on Cyclin E/CDK2 complex at the protein level.
P16 (CDKN2A) protein, an INK4 family member, is a tumor suppressor that plays a significant role in CDK4/6-cyclin D restriction, contributing to G1 arrest through direct binding to CDK4 [34].Along with this, CDKN2A and Rb1 are tumor suppressors that affect the manner of reduction of MDM2 and the proliferation of P53 [35].Baghdadi et al. 2019 failed to ascertain the positive effect of palbociclib on cell cycle inhibition in patients suffering from advanced pancreatic and biliary cancers with CDKN2A loss or mutation [36].As mentioned earlier, this study has found a significant increase in CDKN2A expression in two cell lines.Through the P16 protein, CDKN2A can regulate Rb phosphorylation following CDK4/6 attachment, which can force the cell to stay in the G1 phase and lead to cell cycle arrest.This may have been in line with the palbociclib goal.
The analysis of palbociclib on the expression of signaling pathway genes undertaken here has extended our knowledge to new ways to control and treat breast cancer.However, the study was limited by the lack of information on protein level through western blotting which due to the insufficiencies, a more extensive study is suggested.

Conclusions
Based on our results, palbociclib inhibits cell viability in a dose and time-dependent manner in both MDA-MB-231 and MCF-7 cell lines.Besides, Palbociclib affects the expression of some genes that contributed to cell cycle cascade at the mRNA level and also miR-141 expression as regulator mRNAs in these cell lines.Overexpression of miR-141 results in an interruption in cell proliferation, migration, and invasion.
This study indicates that Palbociclib stops the cell cycle in the G1-S phase by reducing the expression of the CCNE1 gene.The reduction of MDM2 gene expression after treatment with Palbociclib in MCF-7 cell line will lead to cell growth inhibition through P53.Also, by increasing the expression of CDKN2A gene through P16 protein, it forces the cell to stay in G1 phase.